Chondroitin sulphate is a glycosaminoglycan in which glucuronic acid (GlcUA) and an N-acetyl-D-galactosamine (GalNAc) are linearly and alternatively bound by β1-3 linkage and β1-4 linkage respectively to form a linear polysaccharide chain that is sulphated to varying degrees in its GalNAc residues.
It is present in animals, in cartilages and connective tissue, playing an important role in cell adhesion, tissue regeneration, nerve extension and the like.
The production of chondroitin from non-animal sources is an important and desirable step towards the production of non-animal-derived chondroitin sulphate.
The available patent literature describes several methods for the production of non-animal-derived chondroitin.
In addition, several chondroitin synthase genes, derived from both animals and microorganisms, have been cloned and sequenced.
A method for producing chondroitin has been provided by using a recombinant Gram-positive Bacillus bacterium introduced with a chondroitin synthase gene derived from Pasteurella multocida (US 2007/0281342 A1).
A further invention describes a method for producing chondroitin by introducing both the kfoC and kfoA genes, derived from Escherichia coli O5:K4:H4, into a UDP-glucuronic acid-producing bacterium (WO2008/133350).
Another invention describes an in vitro chondroitin synthesis in an enzymatic system comprising both the Escherichia coli O5:K4:H4 chondroitin synthase and the precursors of reaction (US2009/0263867 A1).
It is known that Escherichia coli O5:K4:H4 is able to produce a capsular polysaccharide (the K4 polysaccharide) having the same backbone structure as that of chondroitin, to which fructose residues are linked to the GlcUA residues (see, e.g., N. Volpi, Glycoconj. J., 25:451-457 (2008)). Therefore, the K4 polysaccharide consists of a repeating trisaccharide unit comprising a D-glucuronic acid (GlcUA) moiety and an N-acetyl-D-galactosamine (GalNAc) moiety linked by a β1-3 (GlcUA→GalNAc) and a fructose residue bound to the C3-hydroxyl group of the GlcUA residue. The fructose residues thus constitute branches of the resulting linear polysaccharide.
Removal of the fructose residues has been achieved by hydrolytic treatment in acid conditions (N. Volpi, Electrophoresis, 25, 692-696 (2004)).
Although both the Escherichia coli O5:K4:H4 capsule antigen gene cluster and the metabolic pathways leading to the sugars constituting the K4 linear polysaccharide have been extensively studied, the glycosyl-transferase activity responsible for the addition of the fructose moieties to the linear polysaccharide to give the K4 polysaccharide, has so far not been identified.
The novel feature of the present invention is the direct production of high-molecular weight chondroitin by a recombinant microorganism obtained by inactivating a gene encoding an enzyme responsible for the addition of fructose residues to the chondroitin back-bone thus obviating the need to submit the K4 polysaccharide to the hydrolytic removal of the fructose residues bound to the GlcUA moieties to obtain chondroitin.